Tuberculosis continues to kill two to three million people annually. The incidence of tuberculosis in the United States increased in 1986 after more than 50 years of overall decline. Traditionally, those at risk for clinical disease include the elderly, poor, homeless, and immunocompromised. The latter group now includes HIV infected populations who pose a particularly difficult problem in the Third World where dual infection is prevalent. Recently, great strides were made in adopting the tools of molecular biology to mycobacterial research. Shuttle vectors have been developed to transform DNA into mycobacteria, and to integrate stably integrate foreign genes into the chromosome of mycobacteria. A number of investigators have shown that infection with live mycobacteria is more protective than immunization with dead organisms. These observations led to the hypothesis that the secreted proteins of mycobacteria may be ideal candidates to induce immunoprotective responses. The goal of part I of this proposal is to develop a reporter molecule system in mycobacteria that may be used to screen for secreted or exported proteins. Alkaline phosphatase (phoA) gene fusions, a reporter system that has been extremely useful in identifying exported proteins in other pathogenic organisms, and superoxide dismutase (sod) gene fusions will be evaluated as potential candidates for use as reporter molecules. Each of these potential gene fusion candidates will be ligated to the gene encoding MPB64, a mycobacterial protein known to contain a classical signal sequence, transformed into Mycobacterium smegmatis, and evaluated for secreted alkaline phosphatase (AP) or SOD activity, respectively. Successful expression of either of these reporter enzymes in mycobacteria will allow the screening of hundreds of recombinant colonies for exported proteins. Many previously identified virulence factors from pathogenic organisms are proteins secreted into the membranes, or exported out of the cells where they may interact with the host. The aim of part II of this proposal will be to identify the mechanism of export of SOD from M. tuberculosis; SOD is an immunodominant antigen that has been implicated as a virulence factor, and which does not contain a classical signal sequence. The initial strategy will be to screen a genomic library of M. tuberculosis for its ability to export SOD in M. smegmatis. Neither the SOD from M smegmatis nor from M. tuberculosis is exported when expressed in M smegmatis. These experiments may lead to characterization of unusual mechanisms of protein export in mycobacteria, which may be exploited to allow export of the immunoprotective antigens included in vaccine vectors. Additionally, previously unrecognized exported proteins important in immunoprotection or induction of virulence may be identified.